This invention relates to tubulysin analogs and conjugates thereof, methods for making and using them, and compositions comprising them.
The tubulysins are cytotoxins first isolated from cultures of the myxobacteria Archangium gephyra or Angiococcus disciformis, each producing a different tubulysin mixture (Sasse et al. 2000; Reichenbach et al. 1998). Their crystal structure and biosynthetic pathway have been elucidated (Steinmetz et al. 2004, Ullrich et al. 2009) and their biosynthesis genes have been sequenced (Hoefle et al. 2006b). (Full citations of the references cited herein by first author or inventor and year are listed at the end of this specification.)
The tubulysins belong to a group of antimitotic polypeptides and depsipeptides that includes the phomopsins, the dolastatins, and the cryptophycins (Hamel 2002). Other antimitotic agents are known, for example paclitaxel, the maytansines, and the epothilones. During mitosis, a cell's microtubules reorganize to form the mitotic spindle, a process requiring the rapid assembly and disassembly of the microtubule constituent proteins α- and β-tubulin. Antimitotic agents block this process and prevent a cell from undergoing mitosis. At the molecular level the exact blockage mechanism may differ from one antimitotic agent to another. The tubulysins prevent the assembly of the tubulins into microtubules, causing the affected cells to accumulate in the G2/M phase and undergo apoptosis (Khalil et al. 2006).
The tubulysins have a tetrapeptidyl scaffold consisting of one proteinogenic and three non-proteinogenic amino acid subunits as shown in formula (A): N-methylpipecolinic acid (Mep), isoleucine (Ile), tubuvaline (Tuv), and either tubuphenylalanine (Tup, R′ equals H) or tubutyrosine (Tut, R′ equals OH). Structural variations among the tubulysins (designated A, B, etc.) center around residues R′, R″ and R′″ of formula (A), as shown in Table I. Recently, more naturally occurring tubulysins have been isolated (Chai et al. 2010).
TABLE INaturally Occurring Tubulysins(A) TubulysinR′R″R′′′AOHOC(═O)MeCH2OC(═O)i-BuBOHOC(═O)MeCH2OC(═O)n-PrCOHOC(═O)MeCH2OC(═O)EtDHOC(═O)MeCH2OC(═O)i-BuEHOC(═O)MeCH2OC(═O)n-PrFHOC(═O)MeCH2OC(═O)EtGOHOC(═O)MeCH2OC(═O)CH═CH2HHOC(═O)MeCH2OC(═O)MeIOHOC(═O)MeCH2OC(═O)MeUHOC(═O)MeHVHOHHYOHOC(═O)MeHZOHOHHPretubulysinHHMe
Kaur et al. 2006 studied the antiproliferative properties of tubulysin A and found that it was more potent than paclitaxel and vinblastine and active in xenograft assays against various cancer cell lines. Further, tubulysin A induced apoptosis in cancer cells but not in normal cells and showed significant potential antiangiogenic properties in in vitro assays. The antimitotic properties of other tubulysins have been evaluated and generally compare favorably against those of non-tubulysin antimitotic agents (see, e.g., Balasubramanian et al. 2009, Steinmetz et al. 2004, and Wipf et al. 2004). For these reasons, there is interest in the tubulysins as anti-cancer agents (see, e.g., Domling et al. 2005c and Hamel 2002).
Numerous publications describe efforts directed at the synthesis of tubulysins, including: Balasubramanian et al. 2009, Domling et al. 2006, Hoefle et al. 2003, Neri et al. 2006, Peltier et al. 2006, Sani et al. 2007, Sasse et al. 2007, Shankar et al. 2009, Shibue et al. 2009 and 2010, and Wipf et al. 2004.
Disclosures of tubulysin analogs in which the natural Mep subunit was replaced by an alternative group include Patterson et al. 2007, Wang et al. 2007, Wipf et al. 2010, Balasubramanian et al. 2009, Chai et al. 2011, and Miao et al. 2013.
Several tubulysin analogs where the Ile subunit was replaced by another amino acid have been disclosed: Wipf et al. 2010, Vlahov et al. 2014a, and Zhao et al. 2014b.
Cong et al. 2014 disclose replacement of the R″ acetate group in the Tub subunit with a carbamate group.
Cong et al. 2014 and Cheng et al. 2013 disclose tubulysin analogs in which the R′″ group of the Tuv subunit was replaced with alternative groups. They also disclose replacing the R′ group of the Tup/Tut subunit with an amino group, as a conjugation site.
Balasubramanian et al. 2008 and 2009 disclose tubulysin analogs in which the natural (17S)-Me group in the Tup subunit was replaced by a geminal dimethyl group in conjunction with replacement of the acetate group in the Tuv subunit with a carbonyl group.
Additional disclosures on the preparation of tubulysin analogs or derivatives include: Balasubramanian et al. 2008, Domling 2006, Domling et al. 2005a, Ellman et al. 2013, Hoefle et al. 2001 and 2006a, Pando et al. 2011, Patterson et al. 2008, Raghavan et al. 2008, Richter 2012a, 2012b, and 2012c, Shankar et al. 2013, Shibue et al. 2011, Sreejith et al. 2011, Vlahov et al. 2010a and 2011, Wessjohann et al. 2013, Wipf et al. 2007, Zanda et al. 2013, and Zhao et al. 2014a.
Domling et al. 2005 disclose conjugates of tubulysins with a partner molecule exemplified by polyethylene glycol (PEG). Other disclosures of conjugates of tubulysins are Boyd et al. 2008 and 2010, Jackson et al. 2013, Leamon et al. 2013, Vlahov et al. 2008a, 2008b, 2010b and 2014b, Leamon et al. 2008 and 2010, Reddy et al. 2009, Low et al. 2010, and Zhao et al. 2014a and 2014b. Leung et al. 2002 disclose polyanionic polypeptides that can be conjugated to drugs such as the tubulysins to improve their bioactivity and water solubility. Davis et al. 2008 and Schluep et al. 2009 disclose cyclodextrin based formulations in which tubulysins are covalently attached to a cyclodextrin.